The present invention relates generally to the field of neurological and physiological disfunctions associated with Alzheimer""s Disease. More particularly, the invention is concerned with the identification, isolation and cloning of the gene which when mutated is associated with Alzheimer""s Disease as well as its transcript, gene products and associated sequence information and neighbouring genes. The present invention also relates to methods of diagnosing for and detection of carriers of the gene, Alzheimer Disease diagnosis, gene therapy using recombinant technologies and therapy using the information derived from the DNA, protein, and the metabolic function of the protein.
In order to facilitate reference to various journal articles, a listing of the articles is provided at the end of this specification.
Alzheimer""s Disease (AD) is a degenerative disorder of the human central nervous system characterized by progressive memory impairment and cognitive and intellectual decline during mid to late adult life (Katzman, 1986). The disease is accompanied by a constellation of neuropathologic features principal amongst which are the presence of extracellular amyloid or senile plaques and the neurofibrillary degeneration of neurons. The etiology of this disease is complex, although in some families it appears to be inherited as an autosomal dominant trait. However, even amongst these inherited forms of AD, there are at least three different genes which confer inherited susceptibility to this disease (St George-Hyslop at al., 1990). The xcex54 (Cys112Arg) allelic polymorphism of the Apolipoprotein E (ApoE) gene has been associated with AD in a significant proportion of cases with onset late in life (Saunders et al., 1993; Strittmatter et al., 1993). Similarly, a very small proportion of familial cases with onset before age 65 years have been associated with mutations in the xcex2-amyloid precursor protein (APP) gene (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell et al., 1991; Karlinsky at al., 1992; Mullan et al., 1992). A third locus (AD3) associated with a larger proportion of cases with early onset AD has recently been mapped to chromosome 14q24.3 (Schellenberg at al., 1992; St George-Hyslop et al., 1992; Van Broeckhoven et al., 1992).
Although chromosome 14q carries several genes which could be regarded as candidate genes for the site of mutations associated with AD3 (e.g. cFOS, alpha-1-antichymotrypsin, and cathepsin G), most of these candidate genes have been excluded on the basis of their physical location outside the AD3 region and/or the absence of mutations in their respective open reading frames (Schellenberg, G D et al., 1992; Van Broeckhoven, C et al., 1992; Rogaev et al., 1993; Wong et al., 1993).
There have been several developments and commercial directions in respect of treatment of Alzheimer""s Disease and diagnosis thereof. Published PCT application WO 94 23049 describes transfection of high molecular weight YAC DNA into specific mouse cells. This method is used to analyze large gene complexes, for example the transgenic mice may have increased amyloid precursor protein gene dosage, which mimics the trisomic condition that prevails in Downs Syndrome and the generation of animal models with xcex2-amyloidosis prevalent in individuals with Alzheimer""s Disease. Published international application WO 94 00569 describes transgenic non-human animals harbouring large trans genes such as the trans gene comprising a human amyloid precursor protein gene. Such animal models can provide useful models of human genetic diseases such as Alzheimer""s Disease.
Canadian Patent application 2096911 describes a nucleic acid coding for amyloid precursor protein-cleaving protease, which is associated with Alzheimer""s Disease and Down""s syndrome. The genetic information may be used to diagnose Alzheimer""s disease. The genetic information was isolated from chromosome 19. Canadian patent application 2071105, describes detection and treatment of inherited or acquired Alzheimer""s disease by the use of YAC nucleotide sequences. The YACs are identified by the numbers 23CB10, 28CA12 and 26FF3.
U.S. Pat. No. 5,297,562, describes detection of Alzheimer""s Disease having two or more copies of chromosome 21. Treatment involves methods for reducing the proliferation of chromosome 21 trisomy. Canadian Patent application 2054302, describes monoclonal antibodies which recognize human brain cell nucleus protein encoded by chromosome 21 and are used to detect changes or expression due to Alzheimer""s Disease or Down""s Syndrome. The monoclonal antibody is specific to a protein encoded by human chromosome 21 and is linked to large pyramidal cells of human brain tissue.
By extensive effort and a unique approach to investigating the AD3 region of chromosome 14q, we have isolated, cloned and sequenced the Alzheimer""s related membrane protein (ARMP) gene from within the AD3 region on chromosome 14q24.3. In addition, the direct sequencing of RT-PCR products spanning this 3.0 kb cDNA transcript isolated from affected members of six large pedigrees linked to chromosome 14, has led to the discovery of missense mutations in each of the six pedigrees. These mutations are absent in normal chromosomes. We have established that the ARMP gene is causative of familial Alzheimer""s Disease type AD3. In realizing this link, it is understood that mutations in this gene can be associated with other cognitive, intellectual, or psychological diseases such as cerebral hemorrhage, schizophrenia, depression, mental retardation and epilepsy. These phenotypes are present in these AD families and these phenotypes have been seen in mutations of the APP protein gene. The Amyloid Precursor Protein (APP) gene is also associated with inherited Alzheimer""s Disease. The identification of both normal and mutant forms of the ARMP gene and gene products has allowed for the development of screening and diagnostic tests for ARMP utilizing nucleic acid probes and antibodies to the gene product. Through interaction with the defective gene product and the pathway in which this gene product is involved, gene therapy, manipulation and delivery are now made possible.
Various aspects of the invention are summarized as follows. In accordance with a first aspect of the invention, a purified mammalian polynucleotide is provided which codes for Alzheimer""s related membrane protein (ARMP). The polynucleotide has a sequence which is the functional equivalent of the DNA sequence of ATCC deposit 97124, deposited Apr. 28, 1995. The mammalian polynucleotide may be in the form of DNA, genomic DNA, cDNA, mRNA and various fragments and portions of the gene sequence encoding ARMP. The mammalian DNA is conserved in many species, particularly humans and rodents, especially mice. The souse sequence encoding ARMP has greater than 95% homology with the human sequence encoding the same protein.
Purified human nucleotide sequences which encode mutant ARMP have mutations at nucleotide position i) 685, Axe2x86x92C ii) 737, Axe2x86x92G iii) 986, Cxe2x86x92A, iv) 1105, Cxe2x86x92G and v) 1478, Gxe2x86x92A of Sequence ID No: 1.
The nucleotide sequences encoding ARMP have an alternate splice form in the genes open reading frame. The human cDNA sequence which codas for ARMP has sequence ID No. 1. The mouse sequence which encodes ARMP has sequence ID No. 3. Various DNA and RNA probes and primers may be made from appropriate polynucleotide lengths selected from the sequences. Portions of the sequence also encode antigenic determinants of the ARMP.
Suitable expression vectors comprising the nucleotide sequences are provided along with suitable host cells transfected with such expression vectors.
In accordance with another aspect of the invention, purified mammalian Alzheimer""s related membrane protein is provided. The purified protein has an amino acid sequence encoded by polynucleotide sequence as identified above or the human and mouse sequences of sequence ID No. 2 and sequence ID No. 4. The purified protein may have substitution mutations selected from the group consisting of positions identified in Sequence ID No: 2.
i) M 146L
ii) H 163R
iii) A 246E
iv) L 286V and
v) C 410 Y
Polypeptides of at least six amino acid residues are provided. The polypeptides of six or greater amino acid residues may define antigenic epitopes of the ARMP. Monoclonal antibodies having suitably specific binding affinity for the antigenic regions of the ARMP are prepared by use of corresponding hybridama cell lines. In addition, polyclonal antibodies may be prepared which add suitable specific binding affinities for antigenic regions of the ARMP.
In accordance with another aspect of the invention a bioassay is provided for determining if a subject has a normal or mutant ARMP, where the bioassay comprises
providing a biological sample from the subject
conducting a biological assay on the sample to detect a normal or mutant gene sequence coding for ARMP, a normal or mutant ARMP amino acid sequence, or a normal or defective protein function.
In accordance with another aspect of the invention, a process is provided for producing ARMP comprising culturing one of the above described transfected host cells under suitable conditions, to produce the ARMP by expressing the DNA sequence. Alternatively, ARMP may be isolated from mammalian cells in which the ARMP is normally expressed.
In accordance with another aspect of the invention, a therapeutic composition comprises ARMP and a pharmaceutically acceptable carrier.
In accordance with another aspect of the invention, a recombinant vector for transforming a mammalian tissue cell to express therapeutically effective amounts of ARMP in the cells is provided. The vector is normally delivered to the cells by a suitable vehicle. Suitable vehicles include vaccinia virus, adenovirus, adeno associated virus, retrovirus, liposome transport, neuraltropic viruses and Herpes simplex virus.
In accordance with another aspect of the invention, a method of treating a patient deficient in normal ARMP comprises administering to the patient a therapeutically effective amount of the protein targeted at a variety of patient cells which normally express ARMP. The extent of administration of normal ARMP may be sufficient to override any effect the presence of the mutant ARMP may have on the patient. As an alternative to protein, suitable ligands and therapeutic agents such as small molecules and other drug agents may be suitable for drug therapy.
In accordance with another aspect of the invention an immunotherapy for treating a patient having Alzheimer""s Disease comprises treating the patient with antibodies specific to the mutant ARMP to reduce biological levels or activity of the mutant ARMP in the patient. To facilitate such amino acid therapy, a vaccine composition may be provided for evoking an immune response in a patient of Alzheimer""s Disease where the composition comprises a mutant ARMP and a pharmaceutically acceptable carrier with or without a suitable excipient. Therapy with specific ligands which bind to normal or wild type ARMP of either mutant or wild type and which augments normal function of ARMP in membranes and/or cells or inhibits the bad effect of the mutant.
In accordance with another aspect of the invention, a transgenic animal model for Alzheimer""s Disease has the mammalian polynucleotide sequence with at least one mutation which when expressed results in mutant ARMP in the animal cells and thereby manifests a phenotype. For example, the human Prion gene when overexpressed in rodent peripheral nervous system and muscle cells causes a quite different response in the animal than the human. The animal may be a rodent and is preferably a mouse.
In accordance with another aspect of the invention a transgenic mouse model for Alzheimer""s Disease has the mouse gene encoding ARMP mutated to manifest the symptoms. The transgenic mouse may exhibit symptoms of cognitive memory or behavioural disturbances. In addition or alternatively, the symptoms may appear as cellular tissue disorders such as in mouse liver, kidney spleen or bone marrow.
In accordance with another aspect of the invention, the protein can be used an a starting point for rationale drug design to provide ligands, therapeutic drugs or other types of small chemical molecules.